Pentose Metabolism by Candida utilis

Xylose isomerase, an enzyme isomerizing xylose to xylulose, was produced adaptively when Candida utilis, a fodder yeast, was grown in a medium containing xylose. This experiment was carried out in order to isolate and purify the enzyme, and to clarify properties of the enzyme. As a result, it was revealed that the enzyme could be solubilized by plasmolysis, and was purified by dialysis, salting out with ammonium sulfate, precipitating with acetone, and adsorbing to calcium phosphate gel. The enzyme requires some divalent metal ions for its action and among them manganese ion is the most effective. The enzyme is inhibited particularly by ethylenediamine-tetraacetic acid and has somewhat thermotolerant character.     

Amino Acid Pools and Metabolism During the Cell Division Cycle of Arginine-Grown Candida utilis

Synchronous cultures obtained by isopycnic density gradient centrifugation are used to investigate amino acid metabolism during the cell division cycle of the food yeast Candida utilis. Isotopic labeling experiments demonstrate that the rates of uptake and catabolism of arginine, the sole source of nitrogen, double abruptly during the first half of the cycle, while the cells undergo bud expansion. This is accompanied by a doubling in rate of amino acid biosynthesis, and an accumulation of amino acids. The accumulation probably occurs within the storage pools of the vacuoles. Amino acids derived from protein degradation contribute little to this accumulation. For the remainder of the cell cycle, during cell separation and until the next bud initiation, the rates of uptake and catabolism of arginine and amino acid biosynthesis remain constant. Despite the abrupt doubling in the rate of formation of amino acid pools, their rate of utilization for macromolecular synthesis increases steadily throughout the cycle. The significance of this temporal organization of nitrogen source uptake and amino acid metabolism during the cell division cycle is discussed.     

Hierarchy in Pentose Sugar Metabolism in Clostridium acetobutylicum

Bacterial metabolism of polysaccharides from plant detritus into acids and solvents is an essential component of the terrestrial carbon cycle. Understanding the underlying metabolic pathways can also contribute to improved production of biofuels. Using a metabolomics approach involving liquid chromatography-mass spectrometry, we investigated the metabolism of mixtures of the cellulosic hexose sugar (glucose) and hemicellulosic pentose sugars (xylose and arabinose) in the anaerobic soil bacterium Clostridium acetobutylicum. Simultaneous feeding of stable isotope-labeled glucose and unlabeled xylose or arabinose revealed that, as expected, glucose was preferentially used as the carbon source. Assimilated pentose sugars accumulated in pentose phosphate pathway (PPP) intermediates with minimal flux into glycolysis. Simultaneous feeding of xylose and arabinose revealed an unexpected hierarchy among the pentose sugars, with arabinose utilized preferentially over xylose. The phosphoketolase pathway (PKP) provides an alternative route of pentose catabolism in C. acetobutylicum that directly converts xylulose-5-phosphate into acetyl-phosphate and glyceraldehyde-3-phosphate, bypassing most of the PPP. When feeding the mixture of pentose sugars, the labeling patterns of lower glycolytic intermediates indicated more flux through the PKP than through the PPP and upper glycolysis, and this was confirmed by quantitative flux modeling. Consistent with direct acetyl-phosphate production from the PKP, growth on the pentose mixture resulted in enhanced acetate excretion. Taken collectively, these findings reveal two hierarchies in clostridial pentose metabolism: xylose is subordinate to arabinose, and the PPP is used less than the PKP.     

Regulation of glucose transport in Candida utilis

The transport systems for glucose present in Candida utilis cells, growing in batch and continuous cultures on several carbon sources, have been studied. Two different systems were found: a proton symport and a facilitated diffusion system. The high-affinity symport (Km for glucose about 15 microM) transported one proton per mole of glucose and was partially constitutive, appearing in cells grown on gluconeogenic substrates such as lactate, ethanol and glycerol. It was also induced by glucose concentrations up to 0.7 mM and repressed by higher ones. The level of repression depended on the external glucose concentration at which cells had grown in a way similar to that shown by the maltose-uptake system, so both systems seem to be under a common glucose control. Initial uptake by facilitated diffusion, the only transport system present in cells growing at glucose concentrations higher than 10 mM, showed a complex kinetic dependence on the extracellular glucose concentration. This could be explained either by the presence of at least two different systems simultaneously active, one with a Km around 2 mM and the other with a Km of about 1 M, or by the allosteric or hysteretic behaviour of a single carrier whose apparent Km would oscillate between 2 and 70 mM.     

Extraction of uricase from Candida utilis

Summary Six methods of uricase extraction from Candida utilis were tested and compared. X-press disintegration and sonic oscillation gave the best results.     

Protoplast production from Candida utilis]

The protoplasts of Candida utilis 295 t were produced with the aid of the lytic enzyme from Helix pomatia. If the cell wall of C. utilis 295 t is not treated with SH-compounds (the best effect was found with L-cysteine), it is resistant to the action of the enzyme. The yield of the protoplasts was 100 per cent after 15 minutes of the incubation with the lytic enzyme if the cells were preliminarily treated with L-cysteine. Optimal conditions for the production of the protoplasts are described.     

Heterologous protein secretion by Candida utilis

The yeast Candida utilis (also referred to as Torula) is used as a whole-cell food additive and as a recombinant host for production of intracellular molecules. Here, we report recombinant C. utilis strains secreting significant amounts of Candida antarctica lipase B (CalB). Native and heterologous secretion signals led to secretion of CalB into the growth medium; CalB was enzymatically active and it carried a short N-glycosyl chain lacking extensive mannosylation. Furthermore, CalB fusions to the C. utilis Gas1 cell wall protein led to effective surface display of enzymatically active CalB and of β-galactosidase. Secretory production in C. utilis was achieved using a novel set of expression vectors containing sat1 conferring nourseothricin resistance, which could be transformed into C. utilis, Pichia jadinii, Candida albicans, and Saccharomyces cerevisiae; C. utilis promoters including the constitutive TDH3 and the highly xylose-inducible GXS1 promoters allowed efficient gene expression. These results establish C. utilis as a promising host for the secretory production of proteins.     

产朊假丝酵母(Candida utilis)基因工程研究进展

产朊假丝酵母是生物安全(Generally Recognized as Safe,GRAS)的微生物,也是一种重要的工业微生物。近20年来,随着分子生物学技术的发展,产朊假丝酵母的基因表达系统和基因工程研究及开发应用取得了显著的进展,使得利用该菌表达多种物质成为可能。本文概述了产朊假丝酵母的生物学特点、外源基因表达系统、基因敲除、遗传转化等方面的研究和应用进展。     

Growth of Candida utilis in solid state fermentation

In this work, the growth of the yeast Candida utilis on different solid substrate (wheat bran) and supports (sugarcane bagasse and Amberlite resin) imbibed with a liquid culture medium was studied. Growth was followed by sugars consumption, carbon dioxide production rate (CDPR) and cell count. The results showed the ability of the yeast to grow on the three solid media with fairly good viability and total dextrose consumption in the case of sugarcane bagasse and Amberlite, and partial consumption of wheat bran sugars. After two or three days of culture, a five hundred fold increase in cell population was observed.     

Cobalamins and cobalamin-dependent enzymes in Candida utilis.

Candida utilis has been shown to contain 4.7 pmol of cobalamin per g of wet cell paste. Purification of the cobalamin showed it to be a mixture of methylcobalamin and adenosylcobalamin. Two cobalamin-dependent enzyme systems have been found in the yeast: methylcobalamin-dependent methionine biosynthesis and leucine 2,3-aminomutase. The cobalamin extracted from the yeast is as effective as authentic adenosylcobalamin in stimulating leucine 2,3-aminomutase.     

Protein phosphorylation and trehalase activation in Candida utilis

Abstract Candida utilis cells contain a regulatory trehalase enzyme (280 kDa) which can be activated by cAMP-dependent phosphorylation. A 100-fold purification of this enzyme activity results in the enrichment of a protein band of apparent M _r 70 000 as identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This component is phosphorylated in vivo under conditions in which trehalase activation occurs in whole cells. It is concluded that the trehalase enzyme might be a tetramer, composed of 4 identical 70-kDa subunits.     

Studies on Urate Oxidase of Candida utilis

Some physical and chemical properties of urate oxidase (EC 1.7.3.3) isolated from the cells of Candida utilis were investigated. The molecular weight was estimated to be 1.2 × 10⁵ by the equilibrium sedimentation and gel filtration methods. The isoelectric point was determined as 5.4 by the method of density electrofocusing. The enzyme showed a slight absorption at 410 mμ, and the absorbancy at this wave length was only 3% of that at 280 mμ. Contrary to urate oxidase from swine liver, the enzyme from yeast contained a negligible amount of copper, but it contained iron of nearly one atom per mole of the enzyme protein. The yeast urate oxidase was not inactivated by some chelators. However, it was easily inactivated with certain heavy metal ions such as Hg²⁺, and the inactivated enzyme was reactivated by the addition of thiols, indicating that the enzyme is a sulfhydryl enzyme. The inactivation of the enzyme with urea, on the other hand, was greatly accelerated by the addition of thiols, and some discussion was added to the results obtained.